Life science research has stimulated an enormous increase in the demand for synthetic oligonucleotides over the last decades. A number of methods in molecular biology and DNA-based diagnostics to amplify, detect, analyze and quantify nucleic acids are now dependent on chemically synthesized oligonucleotides which are employed as primers and probes to amplify or to detect nucleic acid targets. Synthetic nucleic acids are also employed as active ingredients in a variety of novel therapeutics. They are used to block the expression of genes through hybridization to messenger RNA (antisense oligonucleotides), to inhibit the transcription of genes through their specific binding to transcription factors (decoy oligonucleotides), to stimulate the immune system (immunostimulatory sequences) and to bind to a variety of protein and other molecular targets due their engineered three-dimensional shape in a highly selective manner (aptamers). A particularly important and promising application is the use of short, double stranded ribonucleotides to invoke RNA interference in order to down regulate individual genes based on their sequence (siRNA). Molecular tagging of industrial products or lifestock, the sequence-directed formation of nanoscale structures and molecular computing are additional important applications of synthetic oligonucleotides. Synthetic nucleic acids therefore represent a highly promising class of molecules which are very likely to have many industrial uses and to positively affect the quality of life.
Synthetic oligonucleotides are prepared through the repeated condensation of nucleosides or oligomeric nucleotide derivatives. Such condensation reactions are termed “coupling reactions”. The most prominent chemical method to perform coupling reactions is the phosphoramidite approach which is displayed in Scheme 1. In this approach, a nucleotide monomer or oligomer phosphoramidite (1) is reacted with a nucleoside monomer or oligonucleotide (2) that comprises a hydroxyl group in the presence of a catalyst, termed “activator”. The reaction product is a phosphorous acid triester (3) that is subsequently oxidized to a phosphoric acid triester (4). The phosphoramidite approach is largely based on developments reported by Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1862, McBride and Caruthers (1983) Tetrahedron Letters 24:245-248, and Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557, and has been reviewed by Beaucage and Iyer (1992) Tetrahedron 48:2223-2311, each of which is incorporated herein by reference in its entirety.
whereinNu1, Nu2=monomeric or oligomeric nucleoside/nucleotide groups;R=phosphate protective group, e.g. β-cyanoethyl; andR′, R″=alkyl groups, e.g. diisopropyl.
Coupling reactions are either performed in solution or with the nucleoside monomer or oligonucleotide (2) being immobilized on a solid support (solid phase oligonucleotide synthesis=SPOS), which is the preferred method for the synthesis of oligonucleotides. In SPOS oligonucleotides are assembled in a cyclical manner, each cycle consisting of a series of three chemical reactions. The first reaction is a deblocking reaction, i.e. the removal of a front-end protective group from the nucleoside or oligonucleotide bound to the support, for example the removal of a dimethoxytrityl protective group. The second reaction is the coupling reaction of a nucleotide monomer or oligomer phosphoramidite to the partially deprotected nucleoside or oligonucleotide on the support in the presence of an activator. The third reaction is the oxidation of the phosphite triester coupling product to a phosphate triester. Optionally, a capping reaction is included in each cycle either directly before or after the oxidation reaction in order to block those support bound nucleosides or oligonucleotides which failed to react in the coupling reaction and to prevent them from further growth in subsequent chain elongation steps.
One of the major methods of preparing phosphoramidites (1) is to react a nucleoside (5) with a phosphitylating agent (6) in the presence of a catalyst, as displayed in Scheme 2. The catalyst applied in this process is termed an “activator” analogous to the catalyst applied in the synthesis of oligonucleotides mentioned above.
whereinNu=monomeric or oligomeric nucleoside/nucleotide group;R=phosphate protective group, e.g. β-cyanoethyl; andR′, R″=alkyl groups, e.g. diisopropyl.
Several compounds have been described as activators for the synthesis of oligonucleotides via the phosphoramidite approach in the technical literature, most of them being either azoles or azolium salts formed from azoles with strong acids, e.g. azolium triflates. The common feature of the described activators is that they represent weak proton acids as well as good nucleophiles (the nucleophile being either the respective compound itself or its conjugated base). Both functions, i.e. providing a weak proton acid and a nucleophile, are deemed important in the mechanism of phosphoramidite coupling reactions as discussed by Dahl et al. (1987) Nucleic Acids Res. 15:1729-43, and Berner et al. (1989) Nucleic Acids Res. 17:853-864, each of which is incorporated herein by reference in its entirety. Activators for the synthesis of oligonucleotides are also applied as activators for the synthesis of phosphoramidites. Ammonium salts of azoles, e.g. the diisopropylammonium salts of 1H-tetrazole or 4,5-dicyanoimidazole, are also used for this purpose.
A number of activators are being marketed commercially for the synthesis of oligonucleotides. Examples of commercially available activators include 1H-tetrazole, described by Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1862, 4,5-dicyanoimidazole (“DCI”), described by Vargeese et al. (1998) Nucleic Acids Res. 26:1046-1050, 5-ethylthio-1H-tetrazole (“ETT”), described by Wright et al. (1993) Tetrahedron Letters 34:3373-3376, and 5-benzylthio-1H-tetrazole (“BTT”), described by Welz and Muller (2002) Tetrahedron Letters 43:795-797. Each of these references is specifically incorporated herein by reference in its entirety.
Other activators that have been described in the literature include 5-(4-nitrophenyl)-1H-tetrazole, described by Froehler and Matteucci (1983) Tetrahedron Letters 24:3171-3174, 5-(3-nitrophenyl)-1H-tetrazole, described by Rao et al. (1993) J. Chem. Soc. Perkin Trans. I 43-55, N-methylanilinium trifluoracetate, described by Fourrey and Varenne (1984) Tetrahedron Letters 25:4511-4514, 2,4-dinitrophenol, described by Dabkowski et al. (2000) Tetrahedron Letters 41:7535-7539, 1-hydroxybenzotriazole, described by Eritja (1990) Tetrahedron 46:721-730, 2,4-dinitrobenzoic acid, described by Reddy and Farooqui., U.S. Pat. No. 5,574,146, benzimidazolium triflate and other azolium salts, e.g. N-phenylimidazolium triflate, described by Hayakawa et al. (1996) J. Org. Chem. 61:7996-7997 and (2001) J. Am. Chem. Soc. 123:8165-8176, 1,2,3-benzotriazole and 5-substituted derivatives thereof, described by Hudson and Cook, U.S. Pat. No. 4,474,948, pyridinium hydrochloride, described by Gryaznov and Letsinger (1992) Nucleic Acids Res. 20:1879-1882, 1-methyl-5-mercaptotetrazole, described by Efimov et al. (1996) Russ. J. Bioorg. Chem. 22:128-130, a combination of pyridinium trifluoracetate and 1-methylimidazole, described by Sanghvi and Manoharan, U.S. Pat. No. 6,274,725, saccharin, described by Sinha and Revell, International Publication No. WO 03/004512, and other compounds. Each of these references is specifically incorporated herein by reference in its entirety.
The activators described to date have certain disadvantages that warrant the search for new and improved activator molecules. For instance, some of the described activators are hygroscopic, such as pyridinium chloride, which requires very strict exclusion of moisture for their storage and handling. The application of such activators in the synthesis of oligonucleotides results in a high risk for synthesis failure due to the great sensitivity of coupling reactions with respect to moisture. This risk is particularly pronounced when a low molar excess of phosphoramidite is applied, for instance in large scale solid phase oligonucleotide synthesis or in coupling reactions which are performed in solution.
Other activators are sensitive to heat and/or mechanical impacts and may cause explosions under such conditions, e.g. 1H-tetrazole and ETT. 1H-Tetrazole tests positive in a test of mechanical sensitivity with respect to shock (“Fallhammer-test”), and is classified as a Category A explosive in Germany. ETT is classified as a Category C explosive in Germany due to its thermal sensitivity. These activators, when used as powders, require special handling and safety procedures during their purification, storage, shipping, use and disposal which increases the cost in routine applications. They also raise safety concerns as they may cause great damage to personnel, equipment and buildings under special conditions, e.g. in case of a fire, when stored in large quantities.
Other activators such as 5-(4-nitrophenyl)-1H-tetrazole (7) have low solubility in acetonitrile, the preferred solvent for coupling reactions, and may crystallize in the lines and valves of automated oligonucleotide synthesis instruments upon slight variations of the environmental temperature, thus blocking the instrument and causing synthesis failures. This undesired phenomenon also occurs with 1H-tetrazole, which is routinely applied at concentrations near its maximum solubility in acetonitrile (appr. 0.45 M at 25° C.).

Other activators, like DCI, may cause precipitation and associated line clogging or valve failure in certain DNA/RNA synthesis instruments due to the incompatibility of solutions of these activators in acetonitrile with other synthesis solutions applied in such instruments. For instance, a mixture consisting of 10 volume percent of a 0.5 M DCI-solution in acetonitrile and 90 volume percent of a 3% solution of trichloroacetic acid in dichloromethane (w/v) becomes cloudy instantaneously after mixing of the two components and a precipitate is deposited from the resulting suspension. It appears likely that such precipitation is caused by the low solubility of DCI in dichloromethane. A solution of 3% trichloroacetic acid in dichloromethane is widely applied in DNA/RNA synthesis instruments for the removal of dimethoxytrityl protective groups in the deblocking reaction of SPOS cycles. The deblocking reaction is immediately followed by the coupling reaction in an SPOS cycle, thus causing a risk of precipitate forming in the machine, because the employed activator solution may come in direct contact with the solution employed in the deblocking reaction.
In the field of phosphoramidite-mediated RNA synthesis, the activators described so far are generally not active enough to promote coupling efficiencies comparable to those observed in DNA synthesis. This phenomenon especially relates to the most widely used RNA amidites, i.e. 2′-O-tert-butyl-dimethylsilyl protected RNA amidites. With existing activators these amidites require longer coupling times at higher activator and/or amidite concentration, but still result in inferior product yields and purity compared to DNA synthesis. Consequently, it is desirable to develop alternative activators that promote the highly efficient synthesis of RNA oligonucleotides.
Although, as discussed above, a variety of activators for the synthesis of oligonucleotides and phosphoramidites have been described, and some of the described activators are commercially available, there is a need to find improved activators that combine the desired features of high activation efficiency and good solubility with easy, safe and economic handling, which lead to superior synthesis results. In particular, the synthesis of RNA oligonucleotides and other oligonucleotides prepared from sterically demanding phosphoramidites requires more efficient activators than are currently available.
The present invention discloses novel methods and activators for the synthesis of oligonucleotides and nucleoside phosphoramidites based on aryl substituted 5-phenyl-1H-tetrazoles wherein at least one of the substituents on the aromatic ring is a perfluoroalkyl substituent. The disclosed activators are highly efficient in coupling DNA-, RNA- and other phosphoramidites, highly soluble, non-hygroscopic and non-hazardous.